Hepacivirus (HCV), pestiviruses and flaviviruses belong to the Flaviviridae family of viruses (Rice, C. M., Flaviviridae: The viruses and their replication. In: Fields Virology, Editors: Fields, B. N., Knipe, D. M., and Howley, P. M., Lippincott-Raven Publishers, Philadelphia, Pa., Chapter 30, 931-959, 1996).
Hepatitis C virus (HCV) is the leading cause of chronic liver disease worldwide. HCV is known to cause at least 80% of post transfusion hepatitis and a substantial proportion of sporadic acute hepatitis. Preliminary evidence also implicates HCV in many cases of “idiopathic” chronic hepatitis, “cryptogenic” cirrhosis, and probably hepatocellular carcinoma unrelated to other hepatitis viruses, such as hepatitis B virus (HBV). A small proportion of healthy persons appear to be chronic HCV carriers, varying with geography and other epidemiological factors. HCV encodes two proteases, a zinc-dependent metalloproteinase, encoded by the NS2-NS3 region, and a serine protease encoded in the NS3 region. These proteases are required for cleavage of specific regions of the precursor polyprotein into mature peptides.
The current standard of care for the treatment of HCV is treatment with interferon or a combination of interferon and ribavirin, although numerous compounds are in clinical trials for other anti-HCV treatments.
Several patents disclose protease inhibitors for the treatment of HCV. U.S. Pat. No. 6,004,933 to Spruce et al. discloses a class of cysteine protease inhibitors for inhibiting HCV. U.S. Pat. No. 5,990,276 to Zhang et al. discloses synthetic inhibitors of hepatitis C virus NS3 protease. The inhibitor is a subsequence of a substrate of the NS3 protease or a substrate of the NS4A cofactor.
Extracts of plants have also been used to treat HCV infections. For example, U.S. Pat. No. 6,056,961 discloses extracts of the plant Hypericum perforatum and pharmaceutical compositions thereof for the treatment of HCV infection. Other U.S. patents disclosing plant extracts for the treatment of HCV infection include: U.S. Pat. No. 5,837,257 to Tsai et al., U.S. Pat. No. 5,725,859 to Omer et al.
The pestivirus genus includes bovine viral diarrhea virus (BVDV), classical swine fever virus (CSFV, also called hog cholera virus) and border disease virus (BDV) of sheep. Moennig V., et al, Adv. Vir. Res. 41:53-98 (1992). Pestivirus infections of domesticated livestock (cattle, pigs, and sheep) cause significant economic losses worldwide (Meyers, G. and Thiel, H.-J., Advances in Virus Research, 47, 53-118, 1996; Moennig V., et al, Adv. Vir. Res. 41:53-98, 1992).
The flavivirus genus includes more than 68 members separated into groups on the basis of serological relatedness (Calisher et al., J. Gen. Virol. 70:3743, 1993). Clinical symptoms vary and include fever, encephalitis, and hemorrhagic fever (Fields Virology, Editors: Fields, B. N., Knipe, D. M., and Howley, P. M., Lippincott-Raven Publishers, Philadelphia, Pa., Chapter 31, 931-959, 1996). Flaviviruses of global concern that are associated with human disease include the dengue hemorrhagic fever viruses (DHF), yellow fever virus, shock syndrome, and Japanese encephalitis virus (Halstead, S. B., Rev. Infect. Dis. 6:251-264, 1984; Halstead, S. B., Science, 239:476-481, 1988; Monath, T. P., New Eng. J. Med., 319:641-643, 1988).
The current standard of care for treatment of Flaviviridae infection is limited to treatment with interferon or a combination of interferon and ribavirin.
One strategy in treating viral infections has been the targeting of viral proteases, which are essential components in the replication of some viruses. Proteases are enzymes, such as pepsin or trypsin, that catalyze the hydrolysis of a protein. The hydrolysis can result in an active protein or a completely processed protein. Alternatively, proteases can simply degrade the protein completely. A nonlimiting list of viruses that encode proteases include: Retroviridae, Picornaviridae, Herpesviridae, Flaviviradae, Coronaviridae, and Togaviridae. The focus on viral proteases has generated significantly effective treatments for viral infections, perhaps most notably in the treatment of viral infections and the success using them in the treatment of human immunodeficiency virus (HIV) infection. Use of protease inhibitors in combination with reverse transcriptase inhibitors is now a preferred treatment for HIV infection.
Protease inhibitors are described in the patent literature. For example, U.S. Pat. No. 6,114,312 discloses and claims a method of inhibiting HIV by combined use of hydroxyurea, a nucleoside analog, and a protease inhibitor. U.S. Pat. No. 5,872,210 to Medabalimi claims and discloses transfrarne peptide inhibitors of viral protease. U.S. Pat. No. 5,945,413 to Tung et al. discloses and claims compounds that inhibit aspartyl proteases. U.S. Pat. No. 6,100,277 to Tucker et al. discloses and claims methods of treating retroviral infections by administering combinations of protease inhibitors.
Piconavirues are one of the largest families of medically important human pathogens and are the major cause of human diseases such as poliomyelitis, acute hepatitis, myocarditis, and the common cold (Wang, Q. M. (1999) Progress In Drug Research 52:199-219). Picornaviruses are small non-enveloped RNA viruses and encode the 3C protease on a single polycistronic mRNA. Enteroviruses and human rhinoviruses are picornaviruses that encode an additional protease, the 2A protease. The viral 2A and 3C proteases are classified as cysteine proteases. The 3C protease has been the target of antiviral agents because it is present in all members of the picomavirus family and makes multiple cleavages on the polyprotein precursor. The catalytic site of the 3C protease is composed of His-Glu-Cys.
Inhibitors of 3C protease can be peptidic or non-peptidic. Peptidic inhibitors include peptide aldehyde and Michael acceptor derivatives. Non-peptidic protease inhibitors include small molecules containing reactive carbonyl groups. Examples of non-peptidic protease inhibitors include β-lactams, isatins, homophthalimides, naphthoquinone-lactol, quinone-like citrinin hydrate, radicinin, and triterpene sulfates (Wang, Q. M. (1999) Progress In Drug Research 52:199-2 19). All but triterpene sulfates inactivate the 3C protease active site nucleophile. Protease inhibitors have been described for treatment of picrornavirus (see U.S. Pat. No. 5,821,331 to Hammond et al. describing compounds and methods for making peptidyl-aldehydes as anti-pircornaviral agents).
Members of the Herpesviridae family of viruses include cytomegalovirus (CMV), herpes simplex virus type 1 (HSV-1), and herpes simplex virus type 2 (HSV-2). Herpesviridae members encode a serine protease that plays an essential role in virus capsid maturation making the protease essential for replication. The CMV capsid protease assemblin contains Ser-His-His in its active site. Benzoxazinone compounds and monocyclic β-lactams have been reported to inhibit assemblin (Abood, N. A. et al. (1997) Bioorg Med. Chem. Lett. 7:2105-2108; Collier, A. C. et al. (1996) N. Engl. J. Med 334:1011-1017). Peptidic inhibitors have also been reported (Patick, A. K. and K. E. Potts (1998) Clinical Microbiology Reviews 11:614-627). U.S. Pat. Nos. 6,008,033 and 6,083,711 to Abdel-MegUid et al. discloses novel herpes protease crystalline structures and methods of identifying inhibitors of these proteases.
The Coronaviridae family includes human respiratory coronavirus and other large, enveloped, plus strand RNA viruses. These viruses cause highly prevalent diseases in humans and animals. Both viral and host proteases process the primary translation product from a polycistronic mRNA. The coronavirus infectious bronchitis virus encodes a trypsin-like protease with His and Cys residues in the catalytic center (Ng, L. F. et al. (2000) Virology 272(1):27-39). A cysteine protease, papain-like protease (PL1pro), of the human coronavirus 229E (HCoV) regulates the expression of the replicase polyproteins, pp1a and ppa1ab, by cleavage between Gly111 and Asn112, far upstream of its own catalytic residue Cys1054 (Herold, J. et al. (1999) J Biol Chem 274(21):14918-25).
Togaviridae include alphaviruses and rubiviruses. Sinbis and Semliki Forest virus are examples of alphaviruses, and rubella virus is the sole member of the rubivirus genus. These viruses are enveloped, plus RNA viruses. Many can be transmitted by mosquitoes. In alphaviruses the genomic RNA serves as the mRNA which is translated into a polyprotein that is co- and posttranslationally cleaved to yield four polypeptides, nsP1, nsP2, nsP3, and nsP4. nsP2 has been identified has containing the protease activity responsible for this cleavage. In the Sinbis virus, the nsPs are translated as two polyproteins, P123 and P1234. P1234 is cleaved at the 3/4 site to yield P123 and nsP4 which is the complex thought to initiate minus-strand synthesis. P123 is cleaved to produce nsP 1, nsP2, and nsP3 which together with nsP4 form the complexes to perform plus-strand RNA synthesis (Schlesinger, S. and M. J. Sclesinger, Togaviridae: The viruses and their replication. in: Fields Virology, Editors: Fields, B. N., Knipe, D. M., and Howley, P. M., Lippincott Raven Publishers, Philadelphia, Pa., Chapter 27, 825-842, 1996) Thus protease activity is critical in Togaviridae replication.
It is an object of the present invention to provide methods and compositions for the treatment of Flaviviridae infection.
It is a specific object of the present invention to provide methods and compositions for the treatment of HCV infection.
It is another object of the present invention to provide methods and compositions for the treatment of flavivirus and pestivirus infections.
It is another object of the present invention to provide methods and compositions for the inhibition of viral proteases.